XBP1 (X-Box-Binding Protein-1)-Dependent O-GlcNAcylation Is Neuroprotective in Ischemic Stroke in Young Mice and Its Impairment in Aged Mice Is Rescued by Thiamet-G

Abstract

Background and purpose: Impaired protein homeostasis induced by endoplasmic reticulum dysfunction is a key feature of a variety of age-related brain diseases including stroke. To restore endoplasmic reticulum function impaired by stress, the unfolded protein response is activated. A key unfolded protein response prosurvival pathway is controlled by the endoplasmic reticulum stress sensor (inositol-requiring enzyme-1), XBP1 (downstream X-box-binding protein-1), and O-GlcNAc (O-linked β-N-acetylglucosamine) modification of proteins (O-GlcNAcylation). Stroke impairs endoplasmic reticulum function, which activates unfolded protein response. The rationale of this study was to explore the potentials of the IRE1/XBP1/O-GlcNAc axis as a target for neuroprotection in ischemic stroke.

Methods: Mice with Xbp1 loss and gain of function in neurons were generated. Stroke was induced by transient or permanent occlusion of the middle cerebral artery in young and aged mice. Thiamet-G was used to increase O-GlcNAcylation.

Results: Deletion of Xbp1 worsened outcome after transient and permanent middle cerebral artery occlusion. After stroke, O-GlcNAcylation was activated in neurons of the stroke penumbra in young mice, which was largely Xbp1 dependent. This activation of O-GlcNAcylation was impaired in aged mice. Pharmacological increase of O-GlcNAcylation before or after stroke improved outcome in both young and aged mice.

Conclusions: Our study indicates a critical role for the IRE1/XBP1 unfolded protein response branch in stroke outcome. O-GlcNAcylation is a prosurvival pathway that is activated in the stroke penumbra in young mice but impaired in aged mice. Boosting prosurvival pathways to counterbalance the age-related decline in the brain's self-healing capacity could be a promising strategy to improve ischemic stroke outcome in aged brains.

Keywords: O-GlcNAc; XBP1; ischemic stroke; neuroprotection; thiamet-G.

Figure 1. Deletion of Xbp1 in forebrain neurons worsens stroke outcome

A) Verification of Xbp1 deletion in the brain of Xbp1^f/f;Emx1-Cre (Xbp1-cKO) mouse. Xbp1-cKO mice were identified by PCR genotyping. Reverse transcription PCR (RT-PCR) analysis on RNA samples prepared from brain cortex was used to confirm deletion of Xbp1 exon 2 in Xbp1-cKO. B) Xbp1-cKO (KO) and Xbp1^f/f littermates (control) mice were subjected to 30 minutes MCAO. After 24 hours of reperfusion, the animals were evaluated for neurologic deficit scores, and then for infarct volume by TTC staining (shown are representative brain slices). C) Control and Xbp1-cKO mice were subjected to permanent MCAO. On day 3 post surgery, the animals were evaluated for infarct volume by TTC staining (shown are representative brain slices). Horizontal bars represent median value of neurologic scores. Infarct volumes are presented as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 2. XBP1s/HBP/O-GlcNAc axis in the brain

A) The hexosamine biosynthetic pathway (HBP) generates UDP-GlcNAc, which is the substrate for O-GlcNAc protein modification. B–D) Overexpression of XBP1s in neurons activates the HBP and O-GlcNAc modification. XBP1s-TG mice (TG) and Camk2a-tTA littermates (control; C) were on regular drinking water for 8 days. B) Expression of XBP1s-regulated genes in cortex of XBP1s-TG mouse brains evaluated by quantitative PCR (means ± SEM; n = 3 per group; *, p < 0.05). C,D) Levels of GFAT1 protein and O-GlcNAc modification were increased in the XBP1s-TG mouse brains.

Figure 3. Activation of O-GlcNAc modification in the ischemic border region after tMCAO is largely Xbp1-dependent

Control (Xbp1^f/f; A) and Xbp1-cKO (B) mice were subjected to 45 minutes MCAO. Three hours later, mouse brains were collected and stained with O-GlcNAc and MAP2 (neuronal marker) antibodies. Dotted lines indicate the ischemic border (loss or attenuated MAP2 immunostaining). Enlarged images of the boxed areas are shown at the bottom. Shown are the representative brain sections from 3 independent experiments. C) Confocal microscopy analysis depicts that O-GlcNAc signal is predominantly present in neurons (MAP2 positive cells) in the ischemic border region. D) Quantification of the fluorescence intensity of O-GlcNAc staining in the boxed areas (as shown in A and B). The relative fluorescence intensity depicts the fold change in the average fluorescence intensity of cells between ipsilateral (ips) and contralateral (con) regions. Six mice (control, n = 3; Xbp1-cKO, n = 3) were used for quantification. Data are presented as mean ± SEM. **, p < 0.01. Scale bar: 20 μm.

Figure 4. Thiamet-G reduces infarct volumes after transient and permanent MCAO in young mice

A) Dose response of global O-GlcNAc levels in mouse brain cortex after thiamet-G or vehicle treatment (C). B) Time course of global O-GlcNAc levels in the brain cortex induced by 30mg/kg thiamet-G or vehicle (C). C) Mice were dosed with 30mg/kg thiamet-G (T-G) or vehicle. After 18 hours, mice were subjected to 45 minutes MCAO. At 24 hours of reperfusion, the animals were evaluated for infarct volume by TTC staining (shown are representative brain slices). D) Mice were subjected to permanent MCAO, and dosed with thiamet-G (30 mg/kg) or vehicle 30 minutes later. On day 3 post surgery, the animals were evaluated for infarct volume by TTC staining (shown are representative brain slices). Infarct volumes are presented as mean ± SEM. **, p < 0.01.

Figure 5. Post-treatment with thiamet-G improves stroke outcome after permanent MCAO in aged mice

A) O-GlcNAc levels in young and aged mouse brains 3 hours after permanent MCAO. Dotted lines indicate ischemic border (loss or attenuated MAP2 immunostaining). Arrows: autofluorescent signals generated by non-perfused blood vessels. B) Quantification of the fluorescence intensity of O-GlcNAc staining. Fluorescence intensities were measured from 15 cells randomly selected from the ischemic border regions (as shown in A) and corresponding contralateral regions for each mouse. Five mice (young, n = 2; aged, n = 3) were used for quantification. C) Brain cortex O-GlcNAc levels were increased similarly in young and aged mice 9 hours after 30 mg/kg thiamet-G (T-G) or vehicle (C). The high molecular weight regions (bracket) were used to quantify O-GlcNAc levels (means ± SEM; n = 3/group). D Neurologic scores and infarct volumes in aged mice dosed with 30 mg/kg thiamet-G or vehicle 30 minutes after pMCAO. Neurologic scores and infarct volumes are presented as median value or mean ± SEM, respectively. Representative TTC-stained brain slices are shown in the right panel. ****, p < 0.0001, **, p < 0.01, *, p < 0.05.